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ISSN Print: 2617-4693
ISSN Online: 2617-4707
IJABR 2018; 2(2): 53-63
www.biochemjournal.com
Received: 15-05-2018
Accepted: 19-06-2018
Maithri Mohan
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Namratha Pai Kotebagilu
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Lohith Mysuru Shivanna
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Shailasree Sekhar
Institution of Excellence,
Vijnana Bhavana, University
of Mysore, Mysuru,
Karnataka, India
Uliyar Vitaldas Mani
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Asna Urooj
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Correspondence
Asna Urooj
Department of Studies in Food
Science and Nutrition,
Manasagangotri, University of
Mysore, Mysuru, Karnataka,
India
Screening of bioactives, anti-oxidant and anti-cancer
potential of a herbal formulation
Maithri Mohan, Namratha Pai Kotebagilu, Lohith Mysuru Shivanna,
Shailasree Sekhar, Uliyar Vitaldas Mani and Asna Urooj
Abstract Medicinal plants have a role in cancer management. The purpose of this study was to develop a herbal adjunct for symptom management in cancer treatment and evaluate its in vitro antioxidant and anti-
cancer activity. Locally available and traditionally used medicinal plants with known anti-cancer properties were selected for development of the formulation. Two variations of the formulation viz., Raw herbal formulation (RHF) and Heat treated formulation (HTF) were prepared. The activity of the variations was compared with Triphala (TRF). The 80% methanol extract of all the samples exhibited higher antioxidant activity and was chosen for screening the anti-cancer ability. The results of GC-MS showed that bioactives having potential anti-cancer effect were identified in HTF with lower probability. However, bioactive components with anti-oxidant, anti-cancer, anti-tumor and cyto-toxic activity were higher in RHF. The morphological studies confirmed apoptotic effect of RHF. Overall, RHF exhibited higher in vitro antioxidant, anti-cancer and cytotoxicity effect.
Keywords: Herbal formulation, Nutraceuticals, HT-29 cell line, cancer, antioxidants, GC-MS, morphology
1. Introduction
Cancer is a complex disease caused due to multiple genetic changes leading to uncontrolled
proliferation of cells of metastatic ability [1]. Cancer occurs due to both external and internal factors. External factors include tobacco chewing/smoking, infectious organisms, unhealthy
diet and lifestyle; whereas internal factors include inherited genetic mutations, hormones and
immune conditions. These factors may act together or in sequence to cause cancer [2]. Studies
by Ferlay et al. 2015 [3] reported that, the three most commonly diagnosed cancers in the
developed countries were prostate, lungs and colorectum among males; and breast,
colorectum and lungs among females. In the developing countries, commonly diagnosed
cancers were lungs, liver and stomach cancer in males and breast, cervix, uterus and lung
cancer in females. By 2030, the global burden of cancer is expected to grow to 21.7 million
new cancer cases and 13 million cancer deaths due to growth and ageing of the population [2].
Apart from the conventional therapies such as chemotherapy and radiotherapy, researchers
are now focusing on novel plant based products with anti-cancer properties [4]. Several
epidemiological studies have indicated protective effect of vegetables and fruits against cancer. In recent years, isolation, identification, characterization, quantification of
phytochemicals and evaluation of their potential benefits to humans has become an important
area of pharmaceutical sciences. Approximately, 30 classes of phytochemicals have been
isolated and listed for their anticancer potential [4].
The nutritional status of a cancer patient may be affected by the tumor and cancer treatment.
Chemotherapy or radiation therapy directed against the tumor is not confined exclusively to
malignant cells; thus, normal tissues may also be affected by therapy and contribute to
specific nutritional problems like nausea, vomiting, diarrhea etc. When combined modality
treatment is given, the nutritional consequences may be magnified [5]. Nutrition Impact
Symptoms (NIS) such as taste and smell alterations, mucositis, nausea, constipation, pain and
shortness of breath seem to occur frequently in patients undergoing cancer treatment [6]. Cancer patients burdened with drug induced toxicity are benefitting from the complementary
and alternative medicines in a hope to find a better cancer management [7].
Alternatives of anticancer molecules of plant based origin have gained huge attention, which
is cost effective and safe in use due to its natural origin. Aggarwal et al. 2006 reported the in
vitro and in vivo anti-cancer ability of cumin and fennel seed via inhibitory effect on
Internat ional Jour nal of Advanced Bioche mis try Rese arch 2018; 2(2): 53-63
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International Journal of Advanced Biochemistry Research
Nuclear Factor-kB (NF-kB) [8]. Savita Dixit et al. 2010
reported that clove possesses cytotoxic effect on cancer
cells, garlic pods have shown to reduce 50% colon cancer
risk in post-menopausal women and neem leaves have shown immunomodulatory, anti-inflammatory, anti-
carcinogenic and potential antioxidant activity which can be
used in colon, stomach, lung, liver, skin, oral, prostate and
breast cancer treatment [9]. Shukla S and Mehta A, 2015
reviewed and reported various plant based anti-cancer
agents with a potential to treat and manage cancer than the
conventional therapies which induce prolonged toxicity [10].
Among the various plants based formulations used
commercially, Triphala (TRF), a herbal powder made from
three primary fruits namely, Indian gooseberry (Phyllanthus
emblica), dried fruit of Terminalia chebula tree and Vibhitaki (Terminalia bellirica) is reported to exhibit high
antioxidant and colon cleansing effect. When the cytotoxic
effects of aqueous extract of TRF was studied in human
breast cancer cell lines (MCF-7), the treated cells were
found to be decreasing with increasing concentrations of
TRF [11]. TRF was used as reference standard in this study
since HT-29 colon cancer cell line was chosen for in vitro
anti-cancer studies. With this background, the study was
conducted with an aim of developing a herbal drink to serve
as an adjunct for cancer management and screening its
potential as antioxidant and anticancer agent.
2. Materials and methods
2.1 Screening of ingredients Considering the availability of ingredients for the
development of the herbal drink, locally available and
traditional used ingredients were selected and screened for
various biological effects documented in literature.
Ingredients such as Garcinia indica (Kokum), Tinospora
cordifolia (Amritaballi), Curcuma amada (Mango Ginger),
Ocimum tenuiflorum (Krishna Tulsi), Mentha piperita
(Mint), Zanthoxylum rhetsa (Sichuan peppers), Cane
jaggery, Borassus flabellifer (Palm jaggery) and Honey were selected for the study and TRF was used as standard.
2.2 Preparation of samples
Leaves of Tinospora cordifolia, Ocimum tenuiflorum and
Mentha piperita were procured from the local market, sorted
and washed. Excess water was removed using paper towels.
Mango ginger was washed thoroughly, the outer skin was
peeled and the root was cut into small pieces.
2.2.1 Preparation of Kokum extract: 400g of kokum rind
was mixed with 2L of water, boiled for 1 hour and strained using a filter. One liter of the filtrate was obtained at the end
of 1 hour and stored at refrigerated temperature until further
use.
2.2.2 Preparation of Sichuan pepper extract: 13g of
Sichuan pepper was weighed and boiled with 350ml of
water for half an hour. The pepper pods were re-extracted in
300ml boiling water until the water reduced to half of its
initial amount. Total of 250ml of the extract was obtained
and stored at refrigeration temperature until further use.
2.3 Development and standardization of herbal
formulation
Since the study aimed at developing a herbal adjunct for
cancer therapy, a formulation was developed aiming to be
used as a herbal drink for cancer patients, using the above
mentioned ingredients at different concentrations. All the
ingredients except kokum and sichaun pepper extracts were
prepared on fresh basis with the necessary preliminary processing, ground to a paste without filtration and freeze
dried (-40ºC) to remove moisture and improve the storage
stability. To study the effect of heat treatment on the
phytochemical composition, the formulation was subjected
to heat treatment (100ºC) (HTF) before freeze drying. The
composition of both the formulations remained the same
except for the processing i.e., heat treatment. The freeze
dried powder of the herbal formulations was used for further
analyses.
2.4 Preparation of Extracts The dehydrated powders of the standard (TRF) and herbal
formulations (RHF and HTF) were extracted using four
solvents viz., aqueous, ethanol, methanol and 80% methanol
in the ratio 1:10 of sample and solvent respectively and
extracted for 24h using a mechanical shaker. The aqueous
extract was freeze dried and solvent extracts were prepared
by flash evaporation. The dried extracts were stored in deep
freezer until further use.
2.5 Chemicals: All the chemicals used were of analytical
grade. Protease, amyloglucosidase, Glucose standards, ß-
carotene standard, 2, 2,-diphenyl-1-picrylhydrazyl (DPPH), Dimethyl Sulfoxide (DMSO), Roswell Park Memorial
Institute Medium (RPMI), Fetal Bovine Serum (FBS),
Penicillin, Streptomycin, Trypsin, 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT), L-glutamine
penicillin streptomycin solution, Triton X-100, Phosphate
Buffered Saline (PBS), Colchicine were purchased from
Sigma-Aldrich, USA. Propidium iodide (PI), 0.25%
Trypsin-EDTA solution, RNase solution (20mg/ml),
Dulbecco’s Modified Eagle Medium (DMEM) and Fetal
Bovine Serum (FBS) were purchased from Himedia
chemicals, India.
2.6 Anti-oxidant Assay
Radical Scavenging Assay (DPPH)
The radical scavenging potential of the herbal formulation
was analyzed using DPPH method described by E. J. Gracia
et al, 2012 [12] and absorbance was read at 517nm. IC50
value was determined using Graph Pad Prism.
Reducing Power Assay (RPA)
Various concentrations of the plant extracts in
corresponding solvents were mixed with phosphate buffer (2.5ml) and potassium ferricyanide (2.5ml). This mixture
was kept at 50°C in water bath for 20 minutes. After
cooling, 2.5ml of 10% trichloroacetic acid was added and
centrifuged at 3000 rpm for 10 minutes. The upper layer of
solution (2.5ml) was mixed with distilled water (2.5ml) and
freshly prepared ferric chloride solution (0.5ml) was added.
The absorbance was measured at 700nm against reagent
blank. Increase in absorbance of the reaction mixture
indicates increase in reducing power [13].
2.7 Screening of Bioactive Compounds using Gas
Chromatography - Mass Spectrometry (GC-MS) analysis
In GC-MS analysis, chromatographic separation was carried
out using the equipment, Thermo GC- Trace Ultra Version: 5.0, Thermo MS DSQ II with Db 35 – MS Capillary
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International Journal of Advanced Biochemistry Research
Standard Non – Polar Column and with a dimension of 30
meters, ID: 0.25mm, film: 0.25µm. Helium was used as
carrier gas with a flow rate of 1.0ml/minute. The oven
temperature was programmed for 70°C and raised to 260°C at a rate of 6°C per minute. About 1µl of the extract was
injected into the instrument. Interpretation on mass spectrum
of GC-MS was conducted using the database of National
Institute Standard and Technology (NIST). The spectrum of
the unknown component was compared with the spectrum
of the known components stored in the NIST library. The
name, molecular weight and molecular formula of the
components of the test materials were ascertained. The
compounds were identified using their retention time,
molecular formula, molecular weight and identified using
the library.
2.8 Cell lines and Culture Colon cancer cells (HT-29) were obtained from National
Centre for Cell Science, Pune, India. The cells were
maintained in the Dulbecco’s Modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS), 1 mM
of sodium bicarbonate, L-glutamine (200 mM),
streptomycin (10 mg/mL), Glucose (25 mM) and penicillin
(10,000 units) (DMEM complete media) at 37 °C in a
humidified 5% CO2 atmosphere. The culture medium was
replaced twice in a week. For the experiments, confluent
cells were trypsinized and plated in 6-well, 12-well and 96-well plates. All cell culture operations were carried out in a
model New Brunswick Galaxy 48 R CO2 incubator from
Eppendorf.
2.9 MTT Assay
The HT-29 cell lines were seeded at a density of 5 × 104
(100 𝜇L/well) in 96-well plates and incubated at 37°C in a
humidified 5% CO2 atmosphere for 24h to form a cell
monolayer. After 24h, the growth medium on the monolayer
was aspirated and treated with 100 𝜇L of 80% methanol extract of TRF, RHF, HTF at various concentrations (0, 500,
1000 and 2000 𝜇g/mL) and Colchicine (320 𝜇g/mL). After
24h treatment, cytotoxicity was tested by MTT [10 𝜇L/well
containing 100 𝜇L of cell suspension; 5 mg/mL of stock in
Phosphate Buffer Saline (PBS)] solution and the plates were
incubated at 37°C for 4h in a 5% CO2 atmosphere. The
supernatants were aspirated from the wells and washed
thrice with PBS. 100 𝜇L of DMSO was added to each well and incubated for 15 min. After incubation, the plates were
gently shaken to solubilize the formazan crystals and
absorbance was measured at 590 nm using multimode plate
reader (Varioskan Flash Top, Thermo Fisher Scientific, Finl
and). The percentage of inhibition (%) was calculated using
the formula below and IC50 values were calculated from log
dose-response curves using Graph Pad Prism software
version 6 for Windows (Graph Pad Software, USA).
Percentage of inhibition (%) =
100 – Test Absorbance at 590nm
Untreated Control Absorbance at 590nm × 100
2.10 Cell Cycle Arrest
The HT-29 cell lines were seeded at a density of 5 × 105 (3
mL/well) in 6-well plates and incubated at 37°C in a
humidified 5% CO2 atmosphere for 24h to form a cell
monolayer. After 24h, the growth medium was aspirated and
treated with 80% methanol extract of RHF (1000 𝜇g/mL)
and Colchicine (320 𝜇g/mL) for 24h. After treatment, the
cells were washed, trypsinized and centrifuged at 1800 rpm
for 8 min. After centrifugation, the supernatant was discarded and the cell pellet was washed twice with PBS.
Further, the cells were re-suspended in PBS (300 𝜇L) and
fixed with 100% ethanol (700 𝜇L) at -20°C for 1 hr. After
fixing, the cells were washed with cold PBS and centrifuged
at 4000 rpm for 10 min at 4°C. The cells were re-suspended
in 1 mL of PBS containing PI (0.05 mg/mL), RNase A (0.05
mg/mL) and Triton X-100 (0.1%), and incubated for 30 min
in the dark at room temperature. Finally, the cells were
sorted in a flow cytometer (Cell Lab Quanta™, SC,
Beckman Coulter, USA).
2.11 Morphological study by Phase contrast Microscopy
HT-29 cells were seeded in a T-25 flask at a density of 2 ×
105 cells/flask and grown for 24hr. After seeding, the cells
were treated with 80% methanol extract of RHF (1000
𝜇g/mL) and Colchicine (320 𝜇g/mL) for 24h, respectively.
After 24h, cell morphology was evaluated using phase
contrast inverted microscope with digital imaging (Axiovert
A1, Zeiss, Germany).
3. Results
3.1 Composition of the herbal formulation
The final composition was standardized by conducting
sensory analysis of the herbal formulation by semi-trained
panel members. Table 1 depicts the composition of herbal
formulation.
Table 1: Composition of herbal formulation
Ingredients Amount
Kokum (Garcinia indica) 5ml
Amritaballi(Tinospora cordifolia) 2g
Mango Ginger (Curcuma amada) 3g
Krishna Tulsi (Ocimum tenuiflorum) 4g
Mint (Mentha piperita) 2g
Sichuan peppers (Zanthoxylum rhetsa) 2ml
Cane jiggery 2g
Palm jaggery (Borassus flabellifer) 3g
Honey 2g
The ingredients used in the herbal formulation have known
antioxidant effect which is justified by literature reports.
Garcinia indica rind has bioactive compounds garcinol and
iso-garcinol which have shown anti-proliferative property in
vitro [14]. Tinospora cordifolia plant extract supplementation
in Swiss albino mouse provided protection against radiation induced alteration in internal mucosa [15]. The bioactive
compound amadaldehyde present in Curcuma amada
exhibited cytotoxicity against the A-549 cell lines [16].
Krishna Tulsi (Ocimum tenuiflorum) leaf extract suppressed
chemically induced hepatomas in rats and tumors in the fore
stomach of mice [17]. Mint (Mentha piperita) leaves provided
protection against oxidative DNA damage associated with
cancer [18]. Sichuan peppers (Zanthoxylum rhetsa) possess
compounds that cause cell apoptosis i.e. by targeting
phosphoinositide-3 kinase (PI-3kinase) pathway required for
cell survival and was also effective in necrosis factor
therapeutics as quoted in a study19. Palm Jaggery (Borassus flabellifer) seed coat inhibited the growth of He La cell lines
(human cell line isolated from cervical cancer patient,
Henrietta Lacks) [20].
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International Journal of Advanced Biochemistry Research
3.2 Antioxidant activity of the herbal extracts
3.2.1. Radical Scavenging Assay (DPPH)
The DPPH assay was carried out in dose dependent manner.
Among the samples, the radical scavenging activity of TRF (which served as standard), was the highest followed by
RHF and HTF samples. Among the extracts of the RHF,
80% methanol showed the highest (67% at 320µg)
antioxidant activity followed by ethanol (64%), aqueous
(64%) and methanol (36%) extract. In the HTF extracts,
80% methanol showed the highest antioxidant activity (38%
at 320µg) followed by ethanol (37%), methanol (29%) and
aqueous (21%) extracts. All the extracts of TRF showed high antioxidant activity (>95% at 320µg) and followed the
order ethanol > methanol > 80% methanol > Aqueous. The
DPPH results of the RHF and HTF extracts as well as that of
the TRF are depicted in table 2.
Table 2: Radical Scavenging Assay (DPPH) of RHF, HTF &TRF Exttracts
Variations % Antioxidant activity
Concentration (µg) 10 20 40 80 160 320
RHF
Aqueous 34.76 24.74 28.11 38.24 29.55 63.8
Ethanol 19.73 16.15 16.87 44.68 64.31 64.41
Methanol 2.27 5.78 5.88 10.95 20.97 35.74
80% Methanol 20.25 25.68 28.64 35.45 65.72 66.85
HTF
Aqueous 14.17 18.51 20.45 12.72 14.89 20.77
Ethanol 11.75 14.09 15.37 17.06 26.97 37.43
Methanol 12.39 15.61 14.33 15.78 19.96 29.3
80% Methanol 15.85 18.25 20.51 22.98 28.85 38.22
TRF
Aqueous 45.52 66.64 94.16 95.28 94.8 95.52
Ethanol 61.76 84.24 95.84 96.32 96.4 97.12
Methanol 46.64 78.8 93.44 96.24 95.6 96.88
80% Methanol 47.68 91.28 95.84 94.96 95.6 95.6
Note: RHF – Raw Herbal Formulation, HTF – Heat treated formulation, TRF - Triphala
3.2.2. Reducing Power Assay
Increase of absorbance in the Reducing Power Assay (RPA)
indicates higher antioxidant activity13. The graphical
representation of the extracts (RHF, HTF and TRF)
subjected to reducing power assay is presented in figure 1.
Among all extracts of the samples, 80% methanol extract
showed maximum reducing power and the result correlated
well with the results observed in the radical scavenging
assay. The standard and variations showed an increasing
trend in the antioxidant activity with increasing
concentrations, however, the antioxidant activity of HTF
was higher followed by RHF and TRF.
Fig 1: Reducing power assay of (A) Triphala; (B) Heat Treated formulation and (C) Raw herbal formulation
3.3 Gas Chromatography - Mass spectrometry (GC-MS)
of herbal extract
Since 80% methanol extract exhibited higher antioxidant
effect in all the samples, it was selected to screen potential
bio-actives by GC-MS analysis and in vitro anti-cancer
studies. The GC-MS results of 80% methanol extracts of
RHF, HTF and TRF are depicted in figures 2, 3 and 4 and
tables 3, 4 and 5 respectively.
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International Journal of Advanced Biochemistry Research
3.3.1. Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Raw Herbal Formulation
Fig 2: Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Raw Herbal Formulation
Table 3: Phyto-components detected in 80% Methanol Extract of RHF
Peak Run Time Compound Name Probability
1 3.96 1,2Dihydroxyfurazan 47.24
5 7.26 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (CAS) 88.67
7 8.53 5-Hydroxymethylfurfural 60.74
8 9.24 4-Hydroxylamino-6-methylpyrimidin-2(1H)-one 5.05
19 21.83 Cyclopropanepentanoic acid, 2-undecyl-, methyl ester, trans- (CAS) 2.19
20 22.61 Hexadecanoic acid (CAS) 63.27
23 29.23 2-Hexadecanol (CAS) 2.44
27 32.07 trans-11-Icosenamide 1.57
The GC-MS results of 80% methanol extract of RHF is depicted in figure 2. From the GC-MS analysis of the RHF
80% methanol extract, compounds having potential anti-
cancer, anti-oxidant, anti-tumorigenic, anti-mutagenic and
cytotoxic activity were identified (table 3). Some of the
compounds with these properties are 1,2Dihydroxyfurazan;
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
(CAS); 5-Hydroxymethylfurfural;4-Hydroxylamino-6-
methylpyrimidin-2(1H)-one; Cyclopropanepentanoic acid 2-
undecyl-,methyl ester, trans- (CAS). Out of these
compounds, 4H-Pyran-4-one, 2, 3-dihydro-3, 5-dihydroxy-6-methyl- (CAS) (88.67%) showed maximum probability
compared to other compounds. It has been reported to
exhibit both antioxidant and cytotoxic effect21. Apart from
these Phyto-compounds 17 different peaks with run time
varying from 5.12 – 31.50 minutes, with molecular weight
from 73 to 207 were detected. However, these compounds
could not be identified as they were not available in the
NIST library.
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International Journal of Advanced Biochemistry Research
Fig 3: Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Heat Treated Herbal Formulation
3.3.2. Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Heat Treated Herbal
Formulation
Table 4: Phyto-components detected in 80% Methanol Extract of HTF
Peak Run Time Compound Name Probability
2 3.76 1,2Dihydroxyfurazan 29.18
5 6.32 Uracil, 1-N-Methyl- 0.78
6 7.24 2-Quinolinamine (CAS) 49.51
7 8.52 5-Hydroxymethylfurfural 75.45
10 10.93 Methyl-eugenol 50.09
17 17.30 3-Deoxy-d-mannoic lactone 74.67
19 21.83 Pentadecanoic acid, 14-methyl-, methyl ester (CAS) 6.14
20 22.62 n-Hexadecanoic acid 45.03
21 25.13 11-Octadecenoic acid, methyl ester 5.18
22 25.98 9-Octadecenoic acid (Z)- (CAS) 12.96
23 26.55 Pentadecanoic acid (CAS) 4.00
25 29.86 11-Octadecenal (CAS) 6.88
26 31.50 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester 6.11
28 34.05 12-Methyl-E,E-2,13-octadecadien-1-ol 4.73
The GC-MS results of 80% methanol extract of HTF is
depicted in figure 3. In HTF 80% methanol extract, the
compounds with potential anti-cancer property that were
identified are 1,2 Dihydroxyfurazan; Uracil, 1-N-Methyl-;5-
Hydroxymethylfurfural; n-Hexadecanoic acid; 11-
Octadecenoic acid; methyl ester, 9-Octadecenoic acid (Z)- (CAS); Pentadecanoic acid (CAS); 11-Octadecenal (CAS);
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester. 5-
Hydroxymethylfurfural (75.45%) which showed highest
probability compared to other compounds and it has been
reported to possess antioxidant as well as anti-cancer
properties22. Apart from these phyto-compounds 14 different
peaks with run time varying from 5.14 – 38.09 minutes,
with molecular weight from 72 to 207 were detected. However these compounds could not be identified as they
were not available in the NIST library.
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International Journal of Advanced Biochemistry Research
3.3.3. Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Triphala
Fig 4: Gas Chromatography and Mass Spectrum Analysis of the 80% Methanol Extract of Triphala
Table 5: Phyto-components detected in 80% Methanol Extract of Triphala (TRF)
Peak Run Time Compound Name Probability
5 7.32 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 10.23
6 7.87 Dodecane (CAS) 21.51
7 8.67 5-Hydroxymethylfurfural 87.28
9 9.87 L-Arginine 11.68
12 11.27 1,2,3-Benzenetriol 90.27
16 15.98 1,2-Cyclopentanedicarboxylic acid, 4-(1,1-dimethylethyl)-, dimethyl ester, (1α,2α,4α)- (CAS) 5.51
18 21.83 Hexadecanoic acid, methyl ester (CAS) 35.66
19 22.6 n-Hexadecanoic acid 61.96
20 25.13 9-Octadecenoic acid (Z)-, methyl ester (CAS) 9.72
21 25.64 Octadecanoic acid, methyl ester (CAS) 33.45
24 32.07 9-Octadecenamide 38.13
28 38.07 Stigmasterol 4.84
30 39.26 4-Piperidineacetic acid, 5-ethylidene-2-[3-(2-hydroxyethyl)-1H-indo l-2-yl]-α-methylene-,
methyl ester, [2S-(2α,4α,5E)]- (CAS) 12.11
The GC-MS results of 80% methanol extract of TRF extract
is depicted in figure 4. TRF was used as reference standard.
80% methanol extract of TRF was screened for bioactives
with anti-cancer properties by GC-MS analysis and the
results showed the presence of compounds such as
Dodecane (CAS); 5-Hydroxymethylfurfural; L-Arginine;
1,2,3-Benzenetriol; 1,2-Cyclopentanedicarboxylic acid,4-
(1,1-dimethylethyl)-dimethyl ester, (1α,2α,4α)- (CAS); Hexadecanoic acid, methyl ester (CAS); n-Hexadecanoic
acid; 9-Octadecenoic acid (Z)-, methyl ester (CAS);
Octadecanoic acid, methyl ester (CAS); Stigmasterol which
have potential anti-cancer properties23. Among these 1, 2, 3-
Benzenetriol (90.27%) reported to exhibit antioxidant effect
showed maximum probability24,25. Apart from these phyto-
compounds 16 different peaks with run time varying from
4.63 – 35.61 minutes, with molecular weight from 72 to 207
were detected. However these compounds could not be
identified as they were not available in the NIST library.
3.4Anti-cancer assay of the herbal extract
3.4.1. MTT Assay MTT assay was carried out on HT-29 colorectal cancer cell
line treated with 80% methanol extract of TRF, RHF and
HTF in dose dependent manner and the percentage growth
inhibition has been depicted in table 8.
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International Journal of Advanced Biochemistry Research
Table 8: Summary of MTT of Test Samples
Extracts Conc. μg/mL OD at 590 nm % Inhibition IC50
Control 0 0.62 0
RHF
500 0.445 28.23
834.5 1000 0.23 62.90
2000 0.16 74.19
HTF
500 0.58 6.45
2335 1000 0.43 30.65
2000 0.355 42.74
TRF
500 0.5 19.35
1492 1000 0.365 41.13
2000 0.265 57.26
Note: RHF – Raw Herbal Formulation, HTF – Heat treated formulation, TRF - Triphala
Results of MTT assay suggested that RHF showed the
lowest IC50 value of 834.5 µg/mL and hence among the
three extracts, RHF was selected for cell cycle arrest and
morphological studies.
3.4.2. Cell cycle arrest
Anti-cancer agents have the capacity to bring about cell
cycle arrest and to induce apoptosis. The human colorectal
adenocarcinoma cell line, (HT-29), was used to investigate the capability of the methanol 80% extract of RHF to induce
apoptosis. Considering the IC50 from the MTT assay, 1000
𝜇g/mL dosage was finalized for the cell cycle analysis. After
24h treatment, 1000 𝜇g/mL dosage of RHF exhibited
significant (P < 0.001) increase in the percentage of cells at
S phase i.e. from 5.670±0.29% to 11.006±0.21% &
8.180±0.07% to 11.006±0.21% as compared to control &
Colchicine 320 𝜇g/mL respectively (Fig.5d). Significant decrease in the proportion of cells in G0/G1 Phase (P <
0.001) was observed.
Fig 5: cell cycle analysis of RHF on HT-29 cells after 24 hours. a) Control; b)RHF 1000 µg/ml; c) Colchicine 320 µg/ml; d) Cell distribution
of HT-29 cells after 24 hour treatment. All values are expressed as mean of triplicates (n=3). Mean values containing different superscript letters a, b, c…,d differ significantly (P˂ 0.001, 0.05)
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International Journal of Advanced Biochemistry Research
3.4.3. Morphological Studies
Fig 6: Morphological changes in HT-29 cell line as observed under phase contrast inverted microscope at 0 and 24 h. (a) Control; (b) Colchicine 320 µg/ml; and (c)RHF 1000 µg/ml., cellular shrinkage (CS), membrane blebbing (MB), nuclear fragmentation (NF), apoptotic
bodies (AB); intact colonies (IC) (magnification 20X).
The HT-29 Cells were treated for 24 hours with the 1000
𝜇g/mL of 80% methanol extract of RHF and Colchicine 320
𝜇g/mL. After exposure for 24hrs the morphological characteristics of apoptosis, such as cellular shrinkage (CS),
membrane blebbing (MB), nuclear fragmentation (NF), and
apoptotic bodies (AB) were examined using phase contrast
inverted microscope. Apoptosis was clearly observed in the
RHF than in the Colchicine 320 µg/ml; which had few intact
colonies along with apoptotic bodies. At higher dose, anti-
cancer activity of RHF was comparable to colchicine.
4. Discussion
Medicinal plants are rich sources of secondary metabolites
such as alkaloids, phenol, cardiac glycosides, flavonoids, tannins and terpenoids determined by gas chromatography
and mass spectrum21. In a study it has been reported that the
activities of some plant constituents with compound nature
of flavonoids, palmitic acid (hexadecanoic acid, ethyl ester
and n-hexadecaonoic acid), unsaturated fatty acid and
linolenic acid (docosatetraenoic acid and octadecatrienoic
acid) have antimicrobial, anti-inflammatory, antioxidant,
hypo-cholesterolemic, cancer preventive, hepato-protective,
anti-arthritic, anti-histimic, anti-eczemic and anti-coronary.
Among the identified phyto-compounds Dodecanoic acid
and n-Hexadecanoic acid have anti-oxidant and anti-microbial activities21. By GC-MS analysis 6 phyto-
constituents in RHF 80% methanol extract, 9 phyto-
constituents in HTF 80% methanol extract and 10 phyto-
constituents in the 80% methanol extract of TRF were
identified, which has potential anti-cancer activity as
reported in literature. Both RHF and HTF extracts showed
the presence of 1, 2 Dihydroxyfurazan and 5-
Hydroxymethylfurfural which have anti-cancer effect and
cytotoxic effect. The probability of 1, 2 Dihydroxyfurazan
was higher in RHF, whereas the probability of 5-Hydroxymethylfurfural was higher in HTF. Comparison
with the commercial herbal supplement TRF showed greater
similarity with the HTF extract but the latter showed lower
probability. Though the composition of both RHF and HTF
sample were the same, a significant difference was observed
in the types of phyto-constituents having anti-cancer effect.
Phyto-constituents having potential anti-cancer effect were
observed in HTF however with lower probability. The
presence of various bioactive components with anti-oxidant,
anti-cancer, anti-tumor and cyto-toxic activity suggests the
possibility of using RHF in the management of cancer. Dose dependent increase in the antioxidant activity was
observed in most of the extracts of the samples with increase
in concentration. This was not observed in aqueous extract
of the formulations. A study showed that 80% methanol
extract of barley seeds showed better antioxidant activity
than 100% methanol extract26. Similar observation was
made in both the RHF and HTF in antioxidant activity.
Plants have a long history of use in treatment of cancer.
Plants are known to possess anticancer activities against
different cancer cell lines. In a study it has been observed
that use of plant extract on cell line (MCF-7) followed by in
vitro anticancer assay with MTT standard, has shown up to 50% growth inhibition27. Similarly, 80% methanol extract of
both RHF and HTF has shown cytotoxic effect via MTT
assay in HT-29 cell line. It was observed that the percentage
growth inhibition in RHF extract was better than the HTF
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International Journal of Advanced Biochemistry Research
extract. This may be due to loss of essential volatile
bioactive compounds, post heat treatment. TRF sample
showed lower percentage growth inhibition than the RHF.
In cell cycle analysis of MDA-MB-231 cell lines after 24 hours treatment with the crude methanol extract of E.
platyloba, it was observed that the percentage of cells in the
S phase significantly increased from 4.58 ± 2.44% to 58.88
± 3.11% as compared to the control28. Similar results were
observed in our study where RHF showed cell cycle arrest at
S phase. The possible mechanism may be due to disruption
of mitochondrial membrane to arrest cells in S phase and
inhibit cell proliferation mediated by RHF. This data is
further supported by morphological studies. Clear apoptosis
was seen after treatment with RHF indicating its apoptotic
ability.
5. Conclusion
Several studies in the past have clearly demonstrated the
beneficial effect of medicinal plants in the alleviation of
human diseases. The therapeutic effects have been attributed
to several compounds present in the plants either solely or in
combination, which have been corroborated by extensive in
vitro and in vivo studies. The present study has
demonstrated the effectiveness of herbal formulation in
curbing cancer cell growth in vitro by induction of
apoptosis. Several bioactives with known anti-cancer effect
have been identified in the formulation through GC-MS analysis. The effect of processing on the profile of bioactive
compound has also been demonstrated. These in vitro
observations pave ways for future clinical studies.
6. Acknowledgements
The authors acknowledge UGC-DRS II project, DOS in
Food Science and Nutrition and Institute of Excellence,
Vijnana Bhavan, Manasagangotri, University of Mysore for
funding and providing infrastructure facilities to conduct the
work.
7. Conflict of interests
Authors declare no conflict of interest.
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